Pyruvate, i.e. a salt of pyruvic acid, is an important intermediate in the pathway of carbohydrate metabolism and fatty acid metabolism in the human and animal body. In order to gain insight in these metabolic pathways, 13C-isotopically enriched pyruvate (hereinafter 13C-pyruvate) has been used to detect metabolites of pyruvate generated in the living body by 13C-NMR.
Further, hyperpolarised 13C-pyruvate has been used as imaging agent in 13C-magnetic resonance (MR) imaging (MRI) and/or spectroscopy (MRS) for in vivo and in vitro 13C-MR studies of metabolic processes in the human and animal body.
The term “hyperpolarised” denotes an enhanced nuclear polarisation of the 13C-nuclei present in the pyruvate molecule. Upon enhancing the nuclear polarisation of the 13C-nuclei, the population difference between excited and ground nuclear spin states of these nuclei is significantly increased and thereby the MR signal intensity is amplified by a factor of hundred and more. When using hyperpolarised 13C-pyruvate, as MR imaging agent, there will be essentially no interference from background signals as the natural abundance of 13C is negligible. Thus the image contrast will be advantageously high. Hyperpolarised 13C-pyruvate may for instance be used as an MR imaging agent for in vivo tumour imaging as described in detail in WO-A-2006/011810 and WO-A-2006/011809. Further, it may be used for assessing the viability of myocardial tissue by MR imaging as described in detail in WO-A-2006/054903. Hyperpolarised 13C-pyruvate can be obtained by for instance dynamic nuclear polarisation (DNP). In this method, 13C-pyruvic acid can be hyperpolarised and is subsequently converted to 13C-pyruvate by using a base for dissolving the solid hyperpolarised 13C-pyruvic acid obtained by DNP. Alternatively, 13C-pyruvate can be directly used in the DNP process in form of certain salts. The production of hyperpolarised 13C-pyruvate is described in detail in WO-A-2006/011809, in WO-A-2007/069909 and WO-A-2007/111515, the latter two applications describing the use of 13C-pyruvates in the described DNP method.
In the body, pyruvate is converted (metabolised) into different compounds: its transamination results in alanine, via oxidative decarboxylation; pyruvate is converted into acetyl-CoA and carbon dioxide (which is further converted to bicarbonate), the reduction of pyruvate results in lactate and its carboxylation in oxaloacetate.
Hyperpolarised 13C-pyruvate which is labelled at the C1-atom, i.e. 13C1-pyruvate is preferably used as MR imaging agent since it has a long T1 relaxation in human full blood (42 s at 37° C.), which allows real-time monitoring and detection of its conversion to hyperpolarised 13C-lactate, hyperpolarised 13C-bicarbonate and hyperpolarised 13C-alanine by 13C-MR/NMR.
Several methods for the production of pyruvic acid are known in the art which can be grossly divided into methods involving the use of microorganisms or enzymes and chemical synthesis.
An example of an enzyme based method for the production of pyruvic acid is the enzymatic oxidation of lactic acid, as for instance described in WO-A-95/00656. Enzymatic oxidation often results in byproducts as reactive hydrogen peroxide is produced during said enzymatic oxidation. Further, an upscale of enzymatic processes to an industrial process level is often problematic or impossible. Examples for methods involving the use of microorganisms for the production of pyruvic acid are for instance described in EP-A-313 850. A disadvantage of these microbiological production processes is that can be difficult and time consuming to separate, isolate and purify pyruvic acid from the complex reaction mixtures, e.g. form complex fermentation broths.
Examples of chemical synthesis for the production of pyruvic acid are largely based on the oxidation of various starting materials like propylene glycol (as described in EP-A-337 246), hydroxyacetone (as disclosed in U.S. Pat. No. 4,247,716) or lactic acid (see for instance JP-A-8183753). However, for the production of isotopically enriched 13C-pyruvic acid, the use of a commercially available isotopically enriched starting material or an isotopically enriched starting material that is obtainable by a straightforward chemical synthesis is greatly preferred.
Further, to be used as hyperpolarised MR imaging agent, 13C1-pyruvic acid has to be of high purity. It is also important that the synthesis can be upscaled since when the compound is used as an MR imaging agent, relatively large amounts of 13C1-pyruvate need to be injected per dose and hence relatively large amounts of 13C1-pyruvic acid need to be polarised. Several methods for the chemical synthesis of isotopically enriched pyruvic acid are known in the art. Seebach et al., Journal of Organic Chemistry 40(2), 1975, 231-237 describe a synthetic route that relies on the protection and activation of a carbonyl-containing starting material as an S,S-acetal, e.g. 1,3-dithian or 2-methyl-1,3-dithian. The dithian is metallated and reacted with a methyl-containing compound and/or 13CO2. By using the appropriate isotopically enriched 13C-compound as outlined in this reference, it is possible to obtain 13C1-pyruvate, 13C2-pyruvate or 13C1,2-pyruvate which may then be converted into the free acid by methods known in the art.
S. H. Anker et al., J. Biol. Chem.176 (1948), 1333-1335 describe a different synthetic route that starts from acetic acid, which is first converted into acetyl bromide and then reacted with Cu13CN. The nitrile obtained is converted into pyruvic acid via the amide. However, the use of toxic cyanides limits the application of this method to small scale production of 13C-labelled pyruvic acid.
We have now surprisingly found a method to produce pyruvic acid and pyruvic acid which is isotopically enriched at the C1-atom, preferably 13C1-pyruvic acid in excellent purity. The method is easily upscaled such that lager amounts of pyruvic acid or isotopically enriched pyruvic acid can be prepared. Apart from 13C-enriched pyruvic acid, the method can also be used to produce 11C-enriched pyruvic acid, 11C1-pyruvic acid. Such a compound can be used as a tracer in positron emission tomography (PET).